CN109054054B - A kind of silk fibroin nanoparticle and preparation method thereof - Google Patents
A kind of silk fibroin nanoparticle and preparation method thereof Download PDFInfo
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Abstract
本发明涉及一种丝素蛋白纳米颗粒及其制备方法，属于纳米材料和高分子生物医用材料技术领域。本发明所述丝素蛋白纳米颗粒为规则球形，不溶于水，粒径在10～1000nm之间，所述纳米颗粒主要为silkⅠ结构，可长期稳定保存。本发明在制备丝素蛋白纳米颗粒过程中，通过调节丝素蛋白浓度和冷冻温度即可直接获得不溶于水的丝素蛋白纳米颗粒。制备方法完全采用水溶液体系，整个制备过程无需添加任何化学试剂，工艺简单，可实现丝素蛋白纳米颗粒的宏量制备，从而彻底解决了现有技术所涉及的化学试剂使用和残留、生物相容性下降以及产量低的问题，对丝素蛋白纳米颗粒的工业化应用具有重要意义。
The invention relates to a silk fibroin nanoparticle and a preparation method thereof, belonging to the technical field of nanomaterials and macromolecular biomedical materials. The silk fibroin nanoparticles of the present invention are regular spherical, insoluble in water, and have a particle size between 10 and 1000 nm. The nanoparticles are mainly of silk I structure and can be stored stably for a long time. In the process of preparing silk fibroin nanoparticles in the present invention, water-insoluble silk fibroin nanoparticles can be directly obtained by adjusting the silk fibroin concentration and freezing temperature. The preparation method completely adopts an aqueous solution system, and the whole preparation process does not need to add any chemical reagents, the process is simple, and the macro-scale preparation of silk fibroin nanoparticles can be realized, thus completely solving the use of chemical reagents and residues and biocompatibility involved in the prior art. The problems of decreased performance and low yield are of great significance for the industrial application of silk fibroin nanoparticles.
Description
Technical Field
The invention belongs to the technical field of nano materials and high-molecular biomedical materials, and particularly relates to silk fibroin nanoparticles and a preparation method thereof.
Background
Silk fibroin is the main component of natural mulberry silk and is secreted by endothelial cells of the silk glands at the back of silkworms. The silk fibroin has high purity, contains no organelles and other biological impurities, and has high biological safety. The silk fibroin has wide application prospect in high and new fields of food, cosmetics, especially biomedical materials and the like due to good biocompatibility, excellent mechanical property and adjustable degradation rate.
After the nano-material is formed, the material has higher specific surface area and chemical activity, and is one of hot spots of research in the field of materials. Protein nanoparticles are attracting attention in the fields of food and cosmetic additives, drug carriers, characteristic material processing and preparation and the like because of good biocompatibility. Methods such as phase separation, salting out, emulsification, electrostatic spraying, etc. have been used to prepare silk fibroin nanoparticles of different sizes. However, the existing preparation method is relatively complex, has low yield and cannot avoid the use of chemical reagents.
The patent ZL 200410014228.2, the patent application CN 106729982A, CN 102417733A and the like continuously report a method for preparing silk fibroin nanoparticles by inducing silk fibroin denaturation by chemical reagents. The silk fibroin nanoparticles reported above cannot avoid the use of chemical reagents, reduce the biocompatibility of the nanoparticles, and are also not beneficial to the loading of bioactive drugs. Patent No. ZL 200410016856.4 reports that the silk fibroin nanospheres are prepared by a freezing-thawing method, but a certain amount of organic solvent such as methanol is required to be added as a denaturant to induce silk fibroin assembly. Patent publication No. CN 101653714A reports that the electrostatic spraying method for preparing silk fibroin particles avoids the use of chemical reagents in the preparation process, but still needs to use organic solvent to induce silk fibroin to be insoluble in water, and the yield is relatively low and the stability of the nanoparticle preparation process under an electrostatic field is poor. Meanwhile, the silk fibroin nano-particles reported above are all stable silk II structures, and the degradation rate is slow.
The invention patent with publication number CN 105832706A provides a silk fibroin nanoparticle with a metastable state structure and a preparation method thereof, the silk fibroin nanoparticle is obtained by cooperatively controlling the conditions of a solvent system, silk fibroin solubility, dissolution, dialysis and centrifugation, the preparation process is complicated, and in addition, a formic acid/salt system is adopted in the preparation process, the molecular weight of the obtained silk fibroin nanoparticle is reduced, and acid and salt are difficult to completely remove.
Before the invention is made, patent application with publication number CN106474482A discloses a silk fibroin nanoparticle, a preparation method and an application thereof, namely, silk fibroin is frozen and induced in liquid nitrogen to assemble the silk fibroin into the nanoparticle, and the nanoparticle is easily induced to be insoluble in water by using an organic solvent after being frozen and dried. The preparation method needs organic solvent induction, repeated cleaning and dispersion, increases the complexity of the preparation process, and obtains the nano-particles with the main structure of the silk II structure and relatively slow degradation.
The present application is particularly proposed based on the above-mentioned drawbacks of the prior art.
Disclosure of Invention
In view of the problems and defects of the prior art, the present invention aims to provide a silk fibroin nanoparticle and a preparation method thereof. The invention adopts a freezing induction self-assembly method, directly obtains silk fibroin nanoparticles which are insoluble in water and mainly have a silk I structure by freezing a silk fibroin aqueous solution at a low temperature and inducing silk fibroin structure transformation by soft freezing, thoroughly avoids the use of chemical reagents, realizes the macro preparation of the nanoparticles and has industrial utilization value.
In order to achieve the first object, the invention provides silk fibroin nanoparticles, which are regular spherical nanoparticles, are insoluble in water, have a particle size of 10-1000 nm, and have a main structure of a silk I structure.
The second object of the present invention is to provide the method for preparing the silk fibroin nanoparticles, which comprises the following steps:
(1) preparing a silk fibroin solution with the mass concentration of 0.01-1.0%, and then placing the silk fibroin solution in liquid nitrogen for freezing until the solution is completely frozen to obtain frozen silk fibroin;
(2) transferring the frozen silk fibroin obtained in the step (1) into a refrigerator for soft freezing treatment, and inducing silk fibroin structure transformation to obtain a water-insoluble silk fibroin nanoparticle frozen body;
(3) directly carrying out freeze drying treatment on the frozen body obtained in the step (2) to obtain the silk fibroin nano-particle powder; or unfreezing the frozen body obtained in the step (2) to obtain silk fibroin nanoparticle suspension with good dispersibility, and then further carrying out freeze drying treatment on the silk fibroin nanoparticle suspension to obtain the silk fibroin nanoparticle powder.
Further, the soft freezing treatment in the step (2) in the technical scheme is freezing treatment for 2-5 days at the temperature of-10-0 ℃.
Further, the thawing in the step (3) of the technical scheme specifically means thawing at 5-60 ℃.
Further, the silk fibroin solution in the step (1) of the technical scheme is a regenerated silk fibroin solution obtained by degumming, dissolving and dialyzing mulberry silk.
The principle of the invention is as follows: the silk fibroin solution is spontaneously assembled to form a nano particle structure in the process of quick freezing, the silk fibroin structure can be induced to be converted into a silk I structure through soft freezing treatment, and the silk fibroin nano particle suspension or powder which is insoluble in water and well dispersed can be obtained through direct thawing or freeze drying.
Compared with the prior art, the silk fibroin nano-particles and the preparation method thereof have the following advantages and remarkable progress:
(1) in the preparation process of the silk fibroin nano particles, the water-insoluble silk fibroin nano particles can be directly obtained only by adjusting the concentration of silk fibroin and the freezing temperature, and the preparation method adopts a full aqueous solution system, and does not need to add any chemical reagent in the whole preparation process, thereby thoroughly solving the problems of the use and the residue of the chemical reagent which are frequently involved in the existing nano particle preparation technology, having higher biological safety and showing obvious advantages in the aspects of food, cosmetics and biomedicine.
(2) The particle size of the Silk fibroin nano-particles prepared by the method is mainly structurally Silk I structure, and the Silk fibroin nano-particles have biodegradability and are faster in degradation rate compared with Silk fibroin nano-particles prepared by a traditional method.
(3) The preparation method is simple, only simple freezing treatment is needed, the process is simple, the yield is high, macroscopic preparation of the fibroin nanoparticles can be realized, and the prepared fibroin nanoparticles can be stably stored for a long time, so that the preparation method has important significance for industrial application of the fibroin nanoparticles.
Drawings
Fig. 1 is a scanning electron microscope image of 10 μm of silk fibroin nanoparticles obtained in example 1 of the present invention;
fig. 2 is a 1 μm scanning electron microscope image of silk fibroin nanoparticles obtained in example 1 of the present invention;
fig. 3 is an XRD spectrum of the silk fibroin nanoparticle obtained in example 1 of the present invention.
Detailed Description
The following is a detailed description of embodiments of the invention. The embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation mode and a specific operation process are given, but the protection scope of the invention is not limited to the following embodiment.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Unless otherwise specified, the temperature at which the silk fibroin solution described in each of the following examples of the present invention was subjected to freezing treatment in liquid nitrogen was-196 ℃.
Example 1
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
soaking 100g of mulberry silk in 5L of 0.05% NaCO3Boiling the solution at 98-100 ℃ for 30min, repeating the boiling for 3 times to degum the silk, and fully washing and drying the degummed silk to obtain pure silk cellulose fiber; mixing silk fibroinVitamin is added into CaCl with the molar ratio of 1:2:82/CH3CH2OH/H2Stirring and dissolving the solution in the ternary O solution at 72 ℃ for 1h to obtain a silk fibroin mixed solution; putting the obtained silk fibroin mixed solution into a dialysis bag, and dialyzing with deionized water for 3 days to obtain a purified silk fibroin solution;
adjusting the mass concentration of the silk fibroin solution to 0.5 wt%, pouring the solution into a metal plate, and placing the metal plate in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at the temperature of-4 ℃ for freezing for 3 days to induce the structure transformation of the silk fibroin;
unfreezing at room temperature of 25 ℃ to obtain silk fibroin nanoparticle suspension with good dispersibility, and then further freeze-drying the prepared silk fibroin nanoparticle suspension to obtain the silk fibroin nanoparticles.
Scanning electron microscope testing is carried out on the finally prepared silk fibroin nano-particles, and the testing results are respectively shown in figure 1 (lower power) and figure 2 (higher power); and simultaneously, XRD (X-ray diffraction) testing is carried out on the obtained silk fibroin nanoparticles, and the detection result is shown in figure 3. As can be seen from figures 1 and 2, the prepared silk fibroin nanoparticles are regular spheres, and the particle size of the particles is 10-1000 nm; as can be seen from fig. 3, the main structure of the silk fibroin nanoparticle is a silk i structure.
Example 2
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
adding degummed mulberry silk cellulose fiber into CaCl with the molar ratio of 1:2:82/CH3CH2OH/H2Stirring and dissolving the solution in the ternary O solution at 60 ℃ for 2 hours to obtain a silk fibroin mixed solution; putting the obtained silk fibroin mixed solution into a dialysis bag, and dialyzing with deionized water for 3 days to obtain a purified silk fibroin solution;
adjusting the mass concentration of the silk fibroin solution to 0.1 wt%, uniformly stirring, pouring into a metal plate, adjusting the thickness of the solution to 3cm, and placing in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at the temperature of-7 ℃ for freezing for 4 days to induce the structure transformation of the silk fibroin;
and (4) performing freeze drying treatment to obtain water-insoluble silk fibroin nanoparticle powder.
Example 3
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
adjusting the mass concentration of the purified mulberry silk fibroin solution to 0.01 wt%, uniformly stirring, pouring into a metal plate, and placing in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at the temperature of-10 ℃ for freezing for 5 days to induce the structure transformation of the silk fibroin;
freeze drying to obtain water insoluble nanometer silk fibroin particle powder;
adding the silk fibroin nanoparticle powder into deionized water, and obtaining well-dispersed silk fibroin nanoparticle suspension again after ultrasonic dispersion.
Example 4
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
adding the degummed mulberry silk cellulose fiber into 9.3mol/L LiBr solution, stirring and dissolving for 2h at 60 ℃ to obtain silk fibroin mixed solution; putting the obtained silk fibroin mixed solution into a dialysis bag, and dialyzing with deionized water to obtain a purified silk fibroin solution;
adjusting the mass concentration of the silk fibroin solution to 0.1 wt%, uniformly stirring, pouring into a metal plate, and placing in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at the temperature of-7 ℃ for freezing for 4 days to induce the structure transformation of the silk fibroin;
and (4) performing freeze drying treatment to obtain water-insoluble silk fibroin nanoparticle powder.
Example 5
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
adding 7mol of degummed mulberry silk cellulose fiber into the degummed mulberry silk cellulose fiberCa (NO) of/L3)2Stirring and dissolving the solution for 2 hours at 80 ℃ to obtain a silk fibroin mixed solution, and dialyzing the mixed solution with deionized water to obtain a purified silk fibroin solution;
adjusting the mass concentration of the silk fibroin solution to 1.0 wt%, uniformly stirring, pouring into a metal plate, and placing in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at the temperature of-1 ℃ for freezing for 2 days to induce the structure transformation of the silk fibroin;
unfreezing at room temperature of 37 ℃ to obtain the silk fibroin nanoparticle suspension with good dispersibility.
Example 6
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
adding degummed mulberry silk cellulose fiber into CaCl with the molar ratio of 1:2:82/CH3CH2OH/H2Stirring and dissolving the solution in the ternary O solution at 60 ℃ for 2 hours to obtain a silk fibroin mixed solution; putting the obtained silk fibroin mixed solution into a dialysis bag, and dialyzing with deionized water for 3 days to obtain a purified silk fibroin solution;
adjusting the mass concentration of the silk fibroin solution to 0.2 wt%, uniformly stirring, pouring into a metal plate, adjusting the thickness of the solution to 3cm, and placing in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at 0 ℃ for freezing for 5 days to induce the structure transformation of the silk fibroin;
unfreezing at room temperature of 5 ℃ to obtain silk fibroin nanoparticle suspension with good dispersibility, and then further freeze-drying the prepared silk fibroin nanoparticle suspension to obtain the silk fibroin nanoparticles.
Example 7
The preparation method of the silk fibroin nanoparticle of the embodiment comprises the following steps:
adding the degummed mulberry silk cellulose fiber into 9.3mol/L LiBr solution, stirring and dissolving for 2h at 60 ℃ to obtain silk fibroin mixed solution; putting the obtained silk fibroin mixed solution into a dialysis bag, and dialyzing with deionized water to obtain a purified silk fibroin solution;
adjusting the mass concentration of the silk fibroin solution to 0.5 wt%, uniformly stirring, pouring into a metal plate, and placing in liquid nitrogen until the solution is completely frozen;
transferring the frozen silk fibroin to a refrigerator at the temperature of-5 ℃ for freezing for 3 days to induce the structure transformation of the silk fibroin;
unfreezing at room temperature of 60 ℃ to obtain silk fibroin nanoparticle suspension with good dispersibility, and then further freeze-drying the prepared silk fibroin nanoparticle suspension to obtain the silk fibroin nanoparticles.
Scanning electron microscope and XRD test were performed on the silk fibroin nanoparticles prepared in the above examples 2 to 7, respectively. Scanning test results show that the silk fibroin nanoparticles prepared in the embodiments 2-7 are regular spheres, and the particle size of the particles is 10-1000 nm; XRD tests can show that the main structure of the prepared silk fibroin nano-particles is a silk I structure.
Claims (2)
1. A silk fibroin nanoparticle, comprising: the nano particles are regular spherical nano particles, are insoluble in water, have particle sizes of 10-1000 nm, and have a main structure of a silk I structure; wherein: the silk fibroin nano-particles are prepared by the following method, comprising the following steps:
(1) preparing a silk fibroin solution with the mass concentration of 0.01-1.0%, and then placing the silk fibroin solution in liquid nitrogen for freezing until the solution is completely frozen to obtain frozen silk fibroin;
(2) transferring the frozen silk fibroin obtained in the step (1) into a refrigerator for soft freezing treatment, and inducing silk fibroin structure transformation to obtain a water-insoluble silk fibroin nanoparticle frozen body; the soft freezing treatment is specifically freezing treatment for 2-5 days at the temperature of-10-0 ℃;
(3) directly carrying out freeze drying treatment on the frozen body obtained in the step (2) to obtain the silk fibroin nano-particle powder; or unfreezing the frozen body obtained in the step (2) to obtain silk fibroin nanoparticle suspension with good dispersibility, and then further carrying out freeze drying treatment on the silk fibroin nanoparticle suspension to obtain silk fibroin nanoparticle powder; the thawing is specifically thawing at 5-60 ℃.
2. The silk fibroin nanoparticle of claim 1, wherein: the silk fibroin solution in the step (1) is regenerated silk fibroin solution obtained by degumming, dissolving and dialyzing mulberry silk.
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| CN114316315B (en) * | 2020-10-09 | 2023-06-27 | 苏州大学 | Method for forming nano-holes on silk fibroin film |
| CN113647409B (en) * | 2021-08-11 | 2023-03-07 | 武汉纺织大学 | Silver-loaded silk fibroin nanoparticle antibacterial agent, preparation method and application |
| CN115054733B (en) * | 2022-07-05 | 2023-06-13 | 武汉纺织大学 | Preparation method and application of silk fibroin/hydroxyapatite composite micro-nano particle material |
| CN116059184B (en) * | 2023-01-18 | 2025-03-11 | 福州大学 | Method for preparing biomolecule nano-particles by freezing |
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| CN102417733A (en) * | 2011-11-11 | 2012-04-18 | 苏州大学 | Preparation method of silk fibroin nanospheres |
| WO2014011644A1 (en) * | 2012-07-09 | 2014-01-16 | Trustees Of Tufts College | High molecular weight silk fibroin and uses thereof |
| CN103923200A (en) * | 2014-03-27 | 2014-07-16 | 苏州丝美特生物技术有限公司 | Silk fibroin freeze-dried powder with large molecular weight |
| JP2017048302A (en) * | 2015-09-01 | 2017-03-09 | 日立化成株式会社 | Method for producing silk fibroin powder |
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